Calcium signaling is essential for numerous biological processes. The NFAT transcription factors, which play key roles in immune cells, bone, heart and skeletal muscle, neurons, glia, pancreas and stem cells, are activated in response to Ca2+ mobilization. NFAT also plays crucial roles in developmental processes and malignant transformation and carcinogenesis. In non-excitable cells, NFAT transcription factors are activated by store-operated Ca2+ entry. Store depletion results in the activation of the ORAI plasma membrane Ca2+ channels by STIM1, a Ca2+ sensor in the endoplasmic reticulum. My sponsor's lab performed a genome-wide RNAi screen in HeLa cells to identify novel components of store-operated Ca2+ entry. The goal of this screen was to further understand the complex pathways associated with Ca2+ signaling. In addition, because current therapeutics target proteins involved in Ca2+ signaling (e.g., cyclosporin A, FK506), the hits from the RNAi screen could be studied as targets of future therapies. Here I propose to further characterize the regulation of Ca2+ signaling by studying four hits from our screen, UEV3, USP13, ArfGAP2, and TMP21, all of which have been implicated in intracellular trafficking. I chose to study these hits because I have previous experience studying proteins and pathways involved in trafficking. We hypothesize that trafficking mechanisms are involved in the movement of STIM1 from internal ER membranes to plasma membrane-apposed ER membranes. In Aim 1 of this proposal, I will capitalize on my strengths in cellular biology and trafficking by investigating the role of UEV3 and USP13 in Ca2+ signaling. I will identify post-translational modifications on STIM that are induced by UEV3/USP13 knockdown. I will also identify other proteins that might form a complex with UEV3 and USP13, and I will examine the localization of UEV3 and USP13 in relation to STIM1 and ORAI1. In Aim 2, I will perform similar experiments with ArfGAP2 and TMP21, two proteins that have been implicated in trafficking. Both proteins will be investigated for their ability to interact with and affect the function and localization of STIM and ORAI. In Ai 3, I will examine store- operated Ca2+ entry and biological function in cells from gene-targeted mice lacking or overexpressing selected trafficking hits from the screen. These experiments will provide me the opportunity to learn new skills and techniques associated with mouse genetic models and the study of primary cell types. The characterization of these proteins will lead to the elucidation of novel pathways that control calcium signaling.